Conventional aquatic feeds are provided in dry, semi-dry, or wet forms. These feeds are either added directly to an aquaculture system or mixed with water prior to use. Conventional feeds rapidly deteriorate in water, with physical decomposition and breakdown of the feed starting immediately upon feed delivery into the culture water. Vulnerable bioactive agents start to decompose when the feed becomes soaked with water, and can then be destroyed by the animal's digestive processes. A barrier of particular significance in the design of feeds and feed additives in all vertebrates, including fish, is the gastrointestinal (GI) tract. Biological, chemical, and physical GI factors, such as varying pH in the GI tract, powerful digestive enzymes, and impermeable GI tract membranes, are all associated with the destruction of biologically or chemically active peptides and other components contained in feed or feed additives. Among the numerous agents which are not typically amenable to common oral administration methods are probiotic bacteria, hormones, polysaccharides, antibiotics, and other organic substances. These agents are rapidly rendered ineffective or are destroyed in the GI tract by acid hydrolysis, enzymes, or other catabolic processes unless adequately protected. A protective approach described in the literature involves coating or top-coating with semi-permeable materials for sustained release formulations (Muhammad et al. 1993). Another approach uses differential water solubility to deliver timed pulse delivery based on water solubility of the coating agents (Amidon and Leesman 1993).
One approach to overcoming some of the disadvantages associated with current feed delivery methods has been the development of microencapsulated diets. EP0237542 (Levine et al. 1987) describes a system where a nutritional component, such as a free amino acid or hormone (see, for example Sache and Bertrand, 1980), was entrapped in a liposome then further encapsulated in a hydrocolloid matrix. The resulting lipogel microcapsules were either stored as a freeze-dried powder or suspended in water containing chloramphenicol. Further, Villamar and Langdon, 1993 described the preparation of complex microcapsules consisting of dietary ingredients and lipid-wall microcapsules embedded in particles of a gelled mixture of alginate and gelatin to obtain a single type of food-particle used to provide suspension feeders with dietary nutrients.
It was also suggested, in WO 87/01587, that microcapsules using liposomes are useful for time-released delivery of materials such as drugs and hormones. These types of microcapsules are based upon phospholipids, which form a membrane around the medication and allow a time release of the medication through this membrane. This type of membrane barrier is fragile, expensive, and difficult to make, and not likely to remain as a discrete microcapsule when combined with other appropriate materials that would be part of a feed for marine animals. Moreover, liposomes are not capable of encapsulating significant amounts of bioactive nutrients.
The microencapsulated feeds described in the art do not solve all of the problems associated with conventional feeds. Production of liposomes and their subsequent encapsulation in a hydrocolloid matrix is a labor-intensive process that adds to the cost of the final feed. Freeze drying of microencapsulated feeds results in oxidation of the lipid component, providing a less desirable feed. Microencapsulated feeds that are stored in a dry state still have some of the same disadvantages as described for dry feeds; that is, they must still be rehydrated and manually introduced into a tank. Furthermore, the microencapsulated feeds described in the prior art have not eliminated the water pollution problems associated with the use of dry feeds.
Several types of starch and polysaccharide polymers have been proposed for use as a matrix for binding bioactive agents then mediating the controlled-release of active agents. Examples of such polymers are poly(vinylpyrrolidone), poly(vinylalcohol), poly(ethylene oxide), cellulose (and cellulose derivatives), silicone and poly(hydroxyethylmethacrylate). Polysaccharide biodegradable matrices are of interest, since the degradation of a natural product, such as starch, occurs naturally in the animal body. A combination of starch and emulsifier has also been envisioned as a method for delivery of materials to foods (Yuan 2000).
Starch and cross-linked starch obtained by treatment with reagents such as epichiorohydrin, phosphorous oxychioride, adipic anhydride, etc. are widely and safely used in the food and pharmaceutical industries with the agreement of the Food and Drug Administration. Several amylolytic enzymes naturally hydrolyze starch. Hence, α-amylase is an endoenzyme specific to α-(1,4)-D-glucopyranoside bonds located within polyglucose chains. The degradation product of starch amylolysis is mainly oligosaccharides, dextrins and maltose. Cross-linked and non-digestible starch has been proposed to enhance the growth of probiotic bacteria in a prebiotic fashion (Brown et al. 2002).
Starch is composed of two distinct fractions: amylose, which is a non-digested fraction containing about 4,000 glucose units, and amylopectin, which is a branched fraction containing about 100,000 glucose units. Hence, amylose and amylopectin differ not only in their chemical structures but also in their digestibility, stability in dilute aqueous solutions, gel texture, and film properties. Micellar crystals held together by hydrogen bonding between amylopectin and amylose are responsible for the integrity of starch granules. When an aqueous suspension of starch is heated to a certain temperature, the hydrogen bonding weakens and the granule swells until collapsing in a process known as “gelatinization.”
The preferred conformation of amylose is a helix of variable dimension; usually it is a left-handed helix with an open core. The consequence of this helical format is that the hydroxyl group located on C6 is most reactive followed by hydroxyl groups on C-3 and finally C-2. Thus, it is possible to introduce a new substituent and chemically modify these —OH groups by, for example, an etherification process, leading to a specifically substituted amylose. The degree of substitution can be adjusted by varying the substituent to amylose ratio (mole of substituent per kg of amylose). For example, different degrees of substitution can be obtained with glycidol, ranging from 0.1 to 10.0. By choosing carefully the substituting agent and the degree of substitution, it is possible to protect the amylose from degradation and to modulate the rate of enzymatic degradation of the polymer. This opens the door to a field of research and development with commercial applications.
Numerous methods of starch gelatinization are well known in the art, including direct or indirect heating of an aqueous dispersion of starch, chemical treatment of such dispersion using strong alkali, or a combination of mechanical and heat treatment. Pre-gelatinized starch is soluble in cold water, suggesting that gelatinization of starch may not be desirable to obtain a controlled-release excipient. However, in accordance with the instant invention, it has been found that the gelatinization of high amylose starch, which can be used as a starting material, permits leaching of the amylose from the starch granules prior to a reaction with a substituted agent. This leaching of amylose provides a controlled time-release property of the instant invention.
In accordance with the instant invention, gelatinization of high amylose starch prior to the addition of substituting agent can be realized by chemical treatment using sodium hydroxide.